Journal: Advanced Science

Article Title: NCOA4 Accelerates Abdominal Aortic Aneurysm Formation by Regulating Macrophage Activation and Ferroptosis via STAT1–CH25H Axis

doi: 10.1002/advs.78079

Figure Lengend Snippet: NCOA4 Transcriptionally Regulates Ch25h in a STAT1‐Dependent Manner. (A) Venn diagram showing the overlap of down‐regulated STAT1 binding peaks and down‐regulated genes from PMs from Ncoa4 −/− mice or Ncoa4 +/+ mice and DEGs from human AAA dataset ( GSE183464 ) and mouse AAA dataset ( GSE17901 ). (B) Relative mRNA expression of indicated genes in PMs from Ncoa4 −/− mice or Ncoa4 +/+ mice at 48 h post‐Ang II administration (n = 6 per group). Data were expressed as the mean ± SEM and analyzed by unpaired t ‐test. (C) IGV view of the Ch25h gene showing sequencing reads from STAT1 CUT & Tag analyses. The promoter regions are highlighted in red. (D) ChIP‐qPCR was performed for STAT1 or NCOA4 and IgG occupancy at Ch25h promoter fragments in indicated groups (n = 6 per group). Data were analyzed by two‐way ANOVA following Tukey's multiple comparisons. (E) Ch25h transcriptional activities were analyzed in HEK293T cells co‐transfected with STAT1 and NCOA4 overexpression plasmids and a luciferase reporter driven by the indicated promoter. pRL‐TK‐renilla plasmid was used as an internal control for transfection efficiency (n = 6 per group). Data were expressed as mean ± SEM and analyzed by one‐way ANOVA following Tukey's multiple comparisons. (F) Western blot of PMs and the quantification (n = 6 per group). Data were analyzed by two‐way ANOVA following Tukey's multiple comparisons. (G) Representative images of IF staining of PMs and the quantification (n = 6 per group). Scale bar indicates 50 µm. Data were expressed as the mean ± SEM and analyzed by unpaired t ‐test. ns indicates no significance; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001. Ang II, angiotensin II; CH25H, cholesterol 25‐hydroxylase; DEGs, differentially expressed genes; IgG, immunoglobulin G; PM, peritoneal macrophage; STAT1, signal transducer and activator of transcription 1.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Binding Assay, Expressing, Sequencing, ChIP-qPCR, Transfection, Over Expression, Luciferase, Plasmid Preparation, Control, Western Blot, Staining